5-Formyluracil and its nucleoside derivatives confer toxicity and mutagenicity to mammalian cells by interfering with normal RNA and DNA metabolism.

Oxidation of the methyl group of thymine yields 5-(hydroxymethyl)uracil (5-hmU) and 5-formyluracil (5-foU) as major products. Whereas 5-hmU appears to have normal base pairing properties, the biological effects of 5-foU are rather poorly characterised. Here, we show that the colony forming ability of Chinese hamster fibroblast (CHF) cells is greatly reduced by addition of 5-foU, 5-formyluridine (5-foUrd) and 5-formyl-2'-deoxyuridine (5-fodUrd) to the growth medium. There are no toxic effects of 5-fodUrd on cells defective in thymidine kinase or thymidylate synthetase, suggesting that the toxicity may be caused by 5-fodUrd phosphorylation and subsequent inhibition of thymidylate synthetase. Whereas 5-fodUrd was the most effective 5-foU derivative causing cell growth inhibition, the corresponding ribonucleoside 5-foUrd was more effective in inhibiting [3H]uridine incorporation in non-dividing rat nerve cells in culture, suggesting that 5-foUrd exerts its toxicity through interference with RNA rather than DNA synthesis. Addition of 5-foU and 5-fodUrd was also found to promote mutagenicity at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus of CHF cells; 5-fodUrd being three orders of magnitude more potent than 5-foU. In contrast, neither 5-hmU nor 5-(hydroxymethyl)-2'-deoxyuridine induced HPRT mutations. The mutation induction indicates that 5-foU will be incorporated into DNA and has base pairing properties different from that of thymine. These results suggest that 5-foU residues, originating from incorporation of oxidised bases, nucleosides or nucleotides or by oxidation of DNA, may contribute significantly to the damaging effects of oxygen radical species in mammalian cells.

Brain hypoplasia caused by exposure to trichlorfon and dichlorvos during development can be ascribed to DNA alkylation damage and inhibition of DNA alkyltransferase repair.

Treatment of pregnant guinea pigs with trichlorfon causes cerebellar hypoplasia in offspring. The most sensitive period for treatment is days 42-47 of gestation, which coincides with the rapid brain growth spurt and with the development of cerebellar granule cells. When rat granule cells were exposed in vitro to trichlorfon and dichlorvos for 24 hours they died, whereas trichloroethanol had no effect. When the cells were exposed to trichlorfon and dichlorvos for 3 hours, only dichlorvos was lethal indicating that the metabolite dichlorvos was more potent than trichlorfon itself. Cultured cerebellar granule cells were also found to be quite sensitive to other DNA-alkylating agents such as methylazoxymethanol and methylmethane sulphonate and to O6-benzylguanine; a potent and specific inhibitor of the DNA alkyltransferase involved in the repair of DNA alkylation damage. The organophosphorous compounds were also found to cause inhibition of the alkyltransferase and the lethal effects of the tested compounds on granule cell culture correlated well with the potency of inhibition. In a bacterial test system for monitoring alkylation effects on the DNA, dichlorvos was demonstrated to have a strong DNA alkylation effect. These results suggest that alkylation of DNA and inhibition of its repair can contribute to the brain hypoplasia observed after exposure to trichlorfon and dichlorvos during brain development.

Inhibition of free radical production or free radical scavenging protects from the excitotoxic cell death mediated by glutamate in cultures of cerebellar granule neurons.

Glutamate kills sensitive neurons through several steps downstream to receptor activation: increased free Ca2+ levels, activation of various enzymes and accumulation of reactive oxygen species (ROS). We have evaluated in a well established model of neuronal cultures the neuroprotective effects of blocking these mechanisms, either singularly or by combining multiple enzyme inhibition and/or ROS scavenging. In vitro cultures of cerebellar granule cells exposed to a toxic concentration of glutamate (100 microM for 15 min in the absence of Mg2+) combined with several pharmacological treatments. Inhibition of nitric oxide synthase (NOS) and phospholipase A2 (PLA2) were effective in decreasing cell death and the combined treatments showed some degree of additivity. By contrast, inhibition of xanthine oxidase (XO) with allopurinol was uneffective. Antioxidants (in particular vitamin e or vitamin E analogs). protected neurons up to more than 50%. A synergistic effect was demonstrated by the combination of vitamin E and C. On the other hand, antioxidants did not increase the protection granted by enzyme inhibitors, suggesting that they act downstream to NOS and PLA2. In conclusion, NOS and PLA2 activated by Ca2+ influx give rise to reactive oxygen species whose deleterious action can be counteracted either by inhibiting these enzymes or by scavenging the excess of free radicals produced by them. Finally, a moderate protection was obtained by blocking protein synthesis with cycloheximide, suggesting a partial contribution of apoptotic mechanisms to the excitotoxic cell death.

Three related brain nuclear receptors, NGFI-B, Nurr1, and NOR-1, as transcriptional activators.

Three related orphan nuclear receptors that are expressed in the brain, NGFI-B, Nurr1, and NOR-1, were studied to compare their function as transcriptional activators. NGFI-B was able to activate (in the absence of added hormone) in CV1 cells both an NGFI-B-responsive luciferase reporter gene (containing eight copies of a response element for NGFI-B upstream of a basal prolactin promoter driving the luciferase gene, NBRE(8)-LUC), a similar thyroid hormone-receptor-responsive reporter gene (TRE(3)-LUC), and a reporter gene with an authentic promoter from a Xenopus vitellogenin gene containing two binding sites for the estrogen receptor (vit-LUC). NGFI-B activated NBRE(8)-LUC and TRE(3)-LUC (but not the vitLUC) with an amino-terminal activation domain. Nurr1 was less promiscuous as a transcriptional activator, activating.the NBRE(8)-LUC better than NGFI-B, but less than NGFI-B at the other reporter genes. NOR-1 activated only the NBRE(8)-LUC reporter gene. These results indicate that closely related nuclear receptors may differentiate between response elements or promoters and that different activation mechanisms exist depending on the promoter. This may contribute to regulation of specificity of target gene expression in the brain.